PROJECT SUMMARY: Sodium-chloride co-transporter (NCC) regulation in the kidney. The Na+-Cl- cotransporter (NCC) in the distal convoluted tubule of the kidney plays a central role in salt, volume and blood pressure homeostasis. The broad, long-term objectives of this project are to understand how NCC regulates extracellular fluid volume and the blood pressure set-point, and to identify novel targets for intervention in the treatment of hypertension. We recently used subcellular fractionation and immuno-electron microscopy to obtain the first in vivo evidence of NCC trafficking in the rat kidney. We discovered that angiotensin II infusion causes an acute redistribution of NCC to the apical plasma membrane while angiotensin converting enzyme (ACE) inhibition causes redistribution into subapical cytoplasmic vesicles. The overall aim of this proposal is to establish the molecular mechanisms by which the renin-angiotensin system (RAS) regulates acute trafficking of NCC. Our preliminary results suggest that angiotensin II-generated reactive oxygen species (ROS) may play a role. Recent studies also suggest that AngII, SPAK/OSR1 and NCC may be sequential components in a phosphorylation cascade, and also that phosphorylation of NCC is associated with its trafficking to the APM. Our central hypothesis is that angiotensin II activates a phosphorylation cascade, perhaps via ROS generation, involving SPAK/OSR1 and NCC, that increases distal tubule Na+ reabsorption by redistributing NCC from intracellular vesicles to the plasma membrane. Aim 1 will test the hypotheses that distal tubule NCC is phosphorylated and redistributed to plasma membrane in response to activation of the renin-angiotensin system and dephosphorylated and redistributed to intracellular membranes when the RAS is inhibited. These hypothesis will be tested in vivo and in renal cortical slices by subcellular fractionation and immuno-electron microscopy of the kidneys using total and phosphospecific NCC antibodies. The role of ROS generation via NADPH oxidase activation in response to AngII and low salt diet will be determined with three distinct inhibitors. Aim 2 will test the hypothesis that activation of the RAS leads to phosphorylation and activation of SPAK/OSR1 and that inhibition of the RAS leads to dephosphorylation. Aim 3 will utilize a recently developed renal epithelial cell model (MCDK) expressing NCC to test the hypothesis that SPAK/OSR1 phosphorylation of NCC increases its activity or abundance in the apical membrane These studies will identify the major molecular components that transduce the signal between the RAS and NCC trafficking, delineate how this pathway is regulated and demonstrate molecular actions of RAS inhibitors used therapeutically to control blood pressure or edema.